A comparison of the folding kinetics of a small, artificially selected DNA aptamer with those of equivalently simple naturally occurring proteins.

The folding of larger proteins generally differs from the folding of similarly large nucleic acids in the number and stability of the intermediates involved. To date, however, no similar comparison has been made between the folding of smaller proteins, which typically fold without well-populated intermediates, and the folding of small, simple nucleic acids. In response, in this study, we compare the folding of a 38-base DNA aptamer with the folding of a set of equivalently simple proteins. We find that, as is true for the large majority of simple, single domain proteins, the aptamer folds through a concerted, millisecond-scale process lacking well-populated intermediates. Perhaps surprisingly, the observed folding rate falls within error of a previously described relationship between the folding kinetics of single-domain proteins and their native state topology. Likewise, similarly to single-domain proteins, the aptamer exhibits a relatively low urea-derived Tanford ß, suggesting that its folding transition state is modestly ordered. In contrast to this, however, and in contrast to the behavior of proteins, ϕ-value analysis suggests that the aptamer's folding transition state is highly ordered, a discrepancy that presumably reflects the markedly more important role that secondary structure formation plays in the folding of nucleic acids. This difference notwithstanding, the similarities that we observe between the two-state folding of single-domain proteins and the two-state folding of this similarly simple DNA presumably reflect properties that are universal in the folding of all sufficiently cooperative heteropolymers irrespective of their chemical details.

Investigation of an anomalously accelerating substitution in the folding of a prototypical two-state protein.

The folding rates of two-state single-domain proteins are generally resistant to small-scale changes in amino acid sequence. For example, having surveyed here over 700 single-residue substitutions in 24 well-characterized two-state proteins, we find that the majority (55%) of these substitutions affect folding rates by less than a factor of 2, and that only 9% affect folding rates by more than a factor of 8. Among those substitutions that significantly affect folding rates, we find that accelerating substitutions are an order of magnitude less common than those that decelerate the process. One of the most extreme outliers in this data set, an arginine-to-phenylalanine substitution at position 48 (R48F) of chymotrypsin inhibitor 2 (CI2), accelerates the protein's folding rate by a factor of 36 relative to that of the wild-type protein and is the most accelerating substitution reported to date in a two-state protein. In order to better understand the origins of this anomalous behavior, we have characterized the kinetics of multiple additional substitutions at this position. We find that substitutions at position 48 in CI2 fall into two distinct classes. The first, comprising residues that ablate the charge of the wild-type arginine but retain the hydrophobicity of its alkane chain, accelerate folding by at least 10-fold. The second class, comprising all other residues, produces folding rates within a factor of two of the wild-type rate. A significant positive correlation between hydrophobicity and folding rate across all of the residues we have characterized at this position suggests that the hydrophobic methylene units of the wild-type arginine play a significant role in stabilizing the folding transition state. Likewise, studies of the pH dependence of the histidine substitution indicate a strong correlation between folding rate and charge state. Thus, mutations that ablate the arginine's positive charge while retaining the hydrophobic contacts of its methylene units tend to dramatically accelerate folding. Previous studies have suggested that arginine 48 plays an important functional role in CI2, which may explain why it is highly conserved despite the anomalously large deceleration it produces in the folding of this protein.

Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains.

Ethylenediamine-tetraacetic acid extracted water-soluble matrix proteins in molluscan shells secreted from the mantle epithelia are believed to control crystal nucleation, morphology, orientation, and phase of the deposited mineral. Previously, atomic force microscopy demonstrated that abalone nacre proteins bind to growing step edges and to specific crystallographic faces of calcite, suggesting that inhibition of calcite growth may be one of the molecular processes required for growth of the less thermodynamically stable aragonite phase. Previous experiments were done with protein mixtures. To elucidate the role of single proteins, we have characterized two proteins isolated from the aragonitic component of nacre of the red abalone, Haliotis rufescens. These proteins, purified by hydrophobic interaction chromatography, are designated AP7 and AP24 (aragonitic protein of molecular weight 7 kDa and 24 kDa, respectively). Degenerate oligonucleotide primers corresponding to N-terminal and internal peptide sequences were used to amplify cDNA clones by a polymerase chain reaction from a mantle cDNA library; the deduced primary amino acid sequences are presented. Preliminary crystal growth experiments demonstrate that protein fractions enriched in AP7 and AP24 produced CaCO(3) crystals with morphology distinct from crystals grown in the presence of the total mixture of soluble aragonite-specific proteins. Peptides corresponding to the first 30 residues of the N-terminal sequences of both AP7 and AP24 were generated. The synthetic peptides frustrate the progression of step edges of a growing calcite surface, indicating that sequence features within the N-termini of AP7 and AP24 include domains that interact with CaCO(3). CD analyses demonstrate that the N-terminal peptide sequences do not possess significant percentages of alpha-helix or beta-strand secondary structure in solution. Instead, in both the presence and absence of Ca(II), the peptides retain unfolded conformations that may facilitate protein-mineral interaction.